Bacillus cereus and application thereof

Abstract

The invention discloses a strain of bacillus cereus and application thereof, the strain is preserved in China General Microbiological Culture Collection Center on November 01, 2023, the preservation number is CGMCC No.1. 60180, and the preservation name is bacillus cereus WHY-3969. The invention also discloses a method for preparing the bacillus cereus. When being applied to the field of agricultural biological prevention and control, the strain can form a biological membrane to be stably colonized at the rhizosphere of a plant, and can generate substances such as IAA and siderophores to promote the growth of the plant. Compared with a blank control potted plant, the bacillus cereus suspension has the advantages that the plant height of the cucumber potted plant is increased by 42.21%, the prevention effect of the cucumber fusarium wilt potted plant is as high as 57.18%, and the bacillus cereus suspension is a biocontrol strain with excellent performance and has good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and particularly relates to a Bacillus cereus WHY-3969 strain and an application thereof. Background Art

[0002] Plant diseases pose a serious threat to global food production and security. The long-standing use of chemical pesticides and fertilizers has exacerbated environmental pollution, bacterial resistance, and soil microbial imbalances. There is an urgent need to identify microbial products that can replace these chemicals. Despite the availability of numerous microbial products, their low colonization potential and short shelf life in the field have led to the discovery of broad-spectrum, highly effective microorganisms from plant-associated microbial communities, such as the rhizosphere and root endophytes, becoming a hot topic for researchers both domestically and internationally. Summary of the Invention

[0003] The present invention provides a strain of Bacillus cereus WHY-3969 and its use in agricultural production. The Bacillus cereus WHY-3969 provided by the present invention was deposited on November 1, 2023, at the General Microbiology Center of the China Culture Collection Committee (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101), with a deposit number of CGMCC No. 1.60180.

[0004] The Bacillus cereus of the present invention is used for preparing biocontrol agents, fertilizers, plant growth agents and / or colonization agents.

[0005] The Bacillus cereus of the present invention is used for preparing medicine for preventing and treating cucumber wilt.

[0006] The invention relates to the use of the Bacillus cereus for preparing IAA and / or siderophore.

[0007] The bacterial suspension, fermentation liquid or fermentation product of the Bacillus cereus of the present invention is used for preparing biocontrol agents, fertilizers, plant growth agents and / or colonization agents.

[0008] The bacterial suspension, fermentation liquid or fermentation product of the Bacillus cereus of the present invention is used for preparing a medicine for preventing and treating cucumber wilt disease.

[0009] The bacterial suspension, fermentation liquid or fermentation product of the Bacillus cereus of the present invention is used for preparing IAA and / or siderogen.

[0010] Optionally, the preparation method of the bacterial suspension and fermentation broth is as follows: first, Bacillus cereus stored at -80°C is streaked on LB solid culture medium and activated by constant temperature culture at 28°C for 24 hours; then the activated strain is inoculated into LB liquid culture medium, shaken and cultured at 160 rpm / min and 28°C for 48 hours, the fermentation broth is centrifuged at 10,000g for 5 minutes, the supernatant is the fermentation broth, and the bacteria are resuspended with an equal amount of sterile water to obtain a bacterial suspension.

[0011] Optionally, the bacterial suspension has a bacterial cell content of 10 8 CFU / mL.

[0012] A biocontrol preparation contains the Bacillus cereus of the present invention.

[0013] The advantages of the present invention are:

[0014] The Bacillus cereus WHY-3969 of the present invention has the ability to form biofilms, produce IAA and siderophores, and the bacterial suspension or fermentation liquid or fermentation product also has the same function. It can stably colonize in soil and plant roots, promote plant growth and prevent and control plant diseases, and is convenient to use, for example, in the preparation of plant disease prevention and growth promotion preparations and biological organic fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following detailed description, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0016] Figure 1 This is the developmental tree of Bacillus cereus WHY-3969 provided in Example 1 of the present invention.

[0017] Figure 2 This is the standard curve of IAA production by Bacillus cereus WHY-3969 provided in Example 3 of the present invention.

[0018] Figure 3 This is the production of the Bacillus cereus WHY-3969 siderophore provided in Example 3 of the present invention.

[0019] Figure 4 The diagram shows the biofilm formation of Bacillus cereus WHY-3969 in different culture media provided in Example 4 of the present invention; A, B, C, and D represent the biofilms of strain WHY-3969 in culture media LBG, LBM, LBGM, and LB, respectively.

[0020] Figure 5 This is the potted control effect of Bacillus cereus WHY-3969 on cucumber wilt provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] The present invention provides a strain of Bacillus cereus WHY-3969 and its application in agricultural production. The Bacillus cereus WHY-3969 provided by the present invention has been deposited in the General Microbiology Center of the China Culture Collection Committee of Microorganisms (referred to as CGMCC, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), Postal Code: 100101, and Deposit Number: CGMCC No.1.60180 on November 1, 2023. When applied to the field of agricultural biological control, it can form a biofilm that stably colonizes in the rhizosphere of plants, and can produce substances such as IAA and siderophores to promote plant growth. The Bacillus cereus suspension increased the height of cucumber potted plants by 42.21% compared with the blank control potted plants, and the prevention effect of cucumber wilt potted plants was as high as 57.18%. It is an excellent biocontrol strain with good development and application prospects.

[0024] The above-mentioned Bacillus cereus WHY-3969 bacterial suspension, fermentation liquid or fermentation product is also within the scope of protection of the present invention.

[0025] The stable colonization of the above-mentioned Bacillus cereus WHY-3969 in soil and plant roots is also within the scope of protection of the present invention.

[0026] The use of the above-mentioned Bacillus cereus WHY-3969 bacterial suspension or fermentation liquid or fermentation product in promoting plant growth is also within the scope of protection of the present invention.

[0027] The use of the above-mentioned Bacillus cereus WHY-3969 bacterial suspension or fermentation liquid or fermentation product in preventing and controlling plant diseases is also within the scope of protection of the present invention.

[0028] The use of the above-mentioned Bacillus cereus WHY-3969 and its bacterial suspension or fermentation liquid or fermentation product in the preparation of plant disease prevention and growth promotion preparations and biological organic fertilizers is also within the scope of protection of the present invention.

[0029] In the above applications, the stable colonization ability is the formation of a biofilm.

[0030] In the above applications, the growth-promoting properties are IAA and siderophores.

[0031] In the present invention, the isolation and purification method of Bacillus cereus WHY-3969 is as follows: 10 g of wheat rhizosphere soil was weighed and added into 200 mL of 0.01 M sterile PBS buffer solution, and the suspension was shaken at room temperature and 180 rpm for 30 min to obtain a rhizosphere soil suspension, which was then gradiently diluted with sterile water to 10 -1 , 10 -2 and 10 -3 , and use a spreading rod to evenly spread it on TYG medium (TYG), whose composition is: 1 g of tryptone, 1 g of yeast extract, 0.5 g of glucose, 6.34 g of KCl, 1.2 g of NaCl, 0.25 g of MgSO4·7H2O, 0.13 g of K2HPO4, 0.22 g of CaCl2·2H2O, 0.17 g of K2SO4, 2.4 g of Na2SO4, 0.5 g of NaHCO3, 0.09 g of Na2CO3, 0.07 g of FeEDTA, 15 g of agar, 1000 mL of distilled water, pH = 7.2-7.4; after constant temperature culture at 28°C for 2 days, pick a single colony for purification culture.

[0032] In the present invention, a Bacillus cereus WHY-3969 bacterial suspension and a de-bacterial fermentation broth are prepared by streaking the strain stored at -80°C onto LB solid culture medium (10g of tryptone, 5g of yeast extract, 10g of NaCl, 20g of agar, 1000mL of distilled water, pH=7.2-7.4) and culturing the culture in a 28°C incubator for 24 hours for activation. The activated strain is then inoculated into a 250mL Erlenmeyer flask containing 100mL of LB liquid culture medium (10g of tryptone, 5g of yeast extract, 10g of NaCl, 1000mL of distilled water, pH=7.2-7.4). The culture was shaken at 160 rpm / min and 28°C for 48 h, and the fermentation broth was centrifuged at 10,000 g for 5 min. The supernatant was the bacterial-free fermentation broth, and the bacteria were resuspended in an equal amount of sterile water to obtain a bacterial suspension.

[0033] Example 1 Isolation, purification and molecular biological identification of strain WHY-3969

[0034] Weigh 10 g of wheat rhizosphere soil and add 200 mL of 0.01 M sterile PBS buffer. Shake at room temperature and 180 rpm for 30 min to obtain a rhizosphere soil suspension. Then, dilute the suspension to 10% with sterile water. -1 , 10 -2 and 10 -3 , and evenly spread it on TYG medium (TYG) with a spreading rod. Its composition is: 1 g tryptone, 1 g yeast extract, 0.5 g glucose, 6.34 g KCl, 1.2 g NaCl, 0.25 g MgSO4·7H2O, 0.13 g K2HPO4, 0.22 g CaCl2·2H2O, 0.17 g K2SO4, 2.4 g Na2SO4, 0.5 g NaHCO3, 0.09 g Na2CO3, 0.07 g FeEDTA, 15 g agar, 1000 mL distilled water, PH = 7.2-7.4; after constant temperature culture at 28°C for 2 days, pick a single colony for purification culture.

[0035] DNA extraction was performed using the column-based bacterial DNA extraction kit from Shanghai Sangon Biotechnology Co., Ltd. The universal bacterial primer pair used was: F27 5'-AGA GTT TGA TCC TGG CTC AGG-3', P1541 5'-AAG GAG GTG GTG ATC CAG CCG CA-3'. Reaction conditions were: denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds, and extension at 72°C for 75 seconds, with 30 cycles in a 50 μL reaction volume. PCR products were verified by agarose gel electrophoresis and sequenced by cloning. The resulting 16S rDNA full sequence was aligned with 16S rDNA sequences obtained from databases such as Genbank, and a phylogenetic tree was constructed using the Mega 6.0 software package.

[0036] The strain WHY-3969 was identified by molecular biological methods. The 16S rDNA base sequence of the strain WHY-3969 is shown in the sequence table. A total of 1437 bases were determined in the 16S rDNA of the strain WHY-3969.

[0037]

[0038] The fragment size of the 16S rDNA sequence obtained by amplification from the genomic DNA of the strain was compared with the 16S rDNA sequence obtained from the Genbank database and the accession number was obtained: WHY-3969 (OR574218.1). Construction of phylogenetic tree: 10 model strains were selected and the Neighbor-Joining method in MEGA 6 software was used for phylogenetic analysis. The constructed phylogenetic tree is shown in Figure 1 shown.

[0039] Example 2 Determination of growth-promoting indexes of strain WHY-3969

[0040] First, strains stored at -80°C were streaked onto LB medium and activated by incubation at 28°C for 24 hours. The activated strains were then inoculated into 100 mL of LB liquid medium in a 250 mL Erlenmeyer flask. 1 mL of L-tryptophan was added to the flask to a final concentration of 100 μg / mL. The culture was then incubated at 28°C and 180 rpm for 5 days, with three biological replicates performed for each strain. Eight mL of fermentation broth was centrifuged at 8000 rpm and 4°C, and the supernatant was collected for quantitative determination of IAA content. For quantitative determination of IAA content, 60 μL of the supernatant was transferred to a 2 mL centrifuge tube, followed by development with 120 μL of Salkowski reagent in the dark. The absorbance of each treatment at 530 nm was measured using a UV spectrophotometer. At the same time, a standard curve of IAA content and absorbance was prepared using gradiently diluted IAA solution (50, 10, 5, 2.5, 1.25, 0.625 and 0 μg / mL), and the IAA production ability of the strain was evaluated based on the standard curve.

[0041] The activated strain was cultured at 28°C for 5 days using the spot inoculation method on an inorganic phosphorus culture medium ((NH4)2SO4 0.5g, NaCl 0.3g, KCl0.3g, MgSO4·7H2O 0.3g, Ca3(PO4)2 25g, FeSO4·7H2O 0.03g, MnSO4·H2O 0.03g, glucose 10g, pH 7.4-7.6). The strain's ability to degrade inorganic phosphorus was evaluated based on whether a transparent zone was formed around the strain and the size of the transparent zone.

[0042] The activated strain was inoculated using the spot inoculation method onto DF medium (2.0 g glucose, 2.0 g gluconic acid, 2.0 g citric acid, 10 mL trace element solution (CaCl2 200 mg, FeSO4·7H2O 200 mg, H3BO3 15 mg, ZnSO4·7H2O 20 mg, Na2MoO4 10 mg, KI 10 mg, NaBr 10 mg, MnCl2 10 mg, COCl2 5 mg, CuCl2 5 mg, AlCl3 2 mg, NiSO4 2 mg, deionized water 1,000 mL), deionized water 990 mL) with 3 mM AC as the sole nitrogen source. DF medium supplemented with 0.2% (w / v) (NH4)2SO4 as the sole nitrogen source served as a positive control, and DF medium served as a negative control. The inoculated plates were incubated at 28°C for 5 days. The strains' ACC production abilities were comprehensively evaluated based on their growth on the three media.

[0043] The activated strain was inoculated on chrome azurol medium (CAS) using the dot inoculation method. The inoculated plates were cultured at 28°C for 7 days. The siderophore production ability of the strain was evaluated based on the appearance and size of the yellow halo around the colony.

[0044] The growth-promoting indexes of the isolated strain WHY-3969, such as IAA production, phosphate solubilization, ACC production and siderophore production, were determined. Figure 2 ), strain WHY-3969 was found to produce 5.97 μg / mL of IAA at 100 μg / mL; it had the ability to produce siderophores (Table 2, Figure 3 ), but it does not have the ability to solubilize phosphorus, potassium, fix nitrogen and produce ACC (Table 2).

[0045] Table 2 Evaluation of growth-promoting properties of strain WHY-3969

[0046]

[0047] Example 3 Biofilm formation assay of strain WHY-3969

[0048] A single colony was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium and cultured overnight at 160 rpm / min and 30°C. 200 μL of LB, LBG (containing 1% glycerol (v / v)), LBM (containing 0.1 mM MnSO4), and LBGM (containing 1% glycerol (v / v) and 0.1 mM MnSO4) medium were added to a 16-well plate. 2 μL of the overnight culture was gently dripped onto the surface of the different media. The 16-well plate was incubated at 30°C for 3 days. Biofilm formation was observed and recorded.

[0049] The results of biofilm assay of strain WHY-3969 showed that the strain could form obvious biofilm in LBG, LBM, LBGM and LB culture media ( Figure 4 ), indicating that the strain can stably colonize in the plant rhizosphere.

[0050] Example 4 Determination of potted plant efficacy of strain WHY-3969 against cucumber wilt

[0051] Cucumber seeds were sown in seedling trays for seedling cultivation. After one week of seedling cultivation, they were transplanted into seedling pots, with one seedling per pot. After transplanting, when the cucumbers grew to the 2-leaf and 1-heart stage, the root irrigation method was used to determine the efficacy. The experimental treatments included: 1) inoculating only 25 mL of cucumber wilt and an equal amount of sterile water (FOC); 2) inoculating 25 mL of cucumber wilt and an equal amount of bacterial suspension (10 8 CFU / mL)(3969-FOC); 3) Inoculate only the bacterial suspension (10 8 CFU / mL) (3969); 4) Inoculate 25 mL of sterile water (CK). Each treatment was replicated four times. One month after inoculation, the disease index, plant height, and fresh weight were calculated, and the control efficacy was calculated based on the disease index.

[0052] Cucumber wilt disease index:

[0053] Level 0: no symptoms;

[0054] Level 1: Less than 1 / 4 of the leaves of the plant show wilting symptoms, the base of the stem is asymptomatic, and the plant grows normally;

[0055] Level 2: 1 / 4-1 / 2 of the leaves of the plant show wilting symptoms, less than 1 / 2 of the stem base turns brown, and the plant becomes dwarfed;

[0056] Level 3: More than 1 / 2 of the leaves of the plant show wilting symptoms, more than 1 / 2 of the stem base turns brown, and the plant is obviously dwarfed;

[0057] Level 4: The whole plant wilts and dies.

[0058]

[0059] Table 3 Disease prevention and growth promotion effect of strain WHY-3969 on cucumber wilt

[0060]

[0061] The strain WHY-3969 had a significant control effect on cucumber wilt, with a control efficiency of 57.18%. In addition, compared with the blank control, the height of cucumber plants treated with the strain WHY-3969 suspension (73.61 cm) was significantly higher than that of the control (42.54 cm) ( Figure 5The fresh weight of cucumber plants treated with strain WHY-3969 was significantly higher than that of the blank control (10.06 g), reaching 20.80 g. This indicates that strain WHY-3969 has a certain growth-promoting effect on cucumber plants (Table 3).

[0062] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings. However, the present disclosure is not limited to the specific details of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.

[0063] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0064] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. A strain of Bacillus cereus, characterized in that The deposit number of the Bacillus cereus is CGMCC No. 1.60180.

2. Use of the Bacillus cereus according to claim 1 in preparing biocontrol agents, fertilizers, plant growth agents and / or colonization agents.

3. Use of the Bacillus cereus according to claim 1 in preparing a drug for preventing and treating cucumber wilt.

4. Use of the Bacillus cereus according to claim 1 for preparing IAA and / or siderophore.

5. Use of the bacterial suspension, fermentation liquid or fermentation product of Bacillus cereus according to claim 1 in preparing biocontrol agents, fertilizers, plant growth agents and / or colonization agents.

6. Use of the bacterial suspension, fermentation liquid or fermentation product of the Bacillus cereus according to claim 1 in preparing a drug for preventing and treating cucumber wilt.

7. Use of the bacterial suspension, fermentation broth or fermentation product of Bacillus cereus according to claim 1 for preparing IAA and / or siderophore.

8. The use according to any one of claims 5 to 7, characterized in that: The preparation method of the bacterial suspension and fermentation liquid is as follows: First, Bacillus cereus stored at -80°C was streaked onto LB solid medium and incubated at 28°C for 24 h for activation; The activated strain was then inoculated into LB liquid culture medium and cultured at 160 rpm / min and 28°C for 48 h. The fermentation broth was centrifuged at 10,000 g for 5 min. The supernatant was the fermentation broth, and the bacteria were resuspended with an equal amount of sterile water to obtain a bacterial suspension.

9. The use according to claim 8, characterized in that The bacterial suspension has a bacterial content of 10 8 CFU / mL.

10. A biocontrol agent, characterized in that: The biocontrol preparation contains the Bacillus cereus according to claim 1.

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